Ca restores redox homeostasis and stress response
As shown in Table 1, a plenty of proteins involved in redox homeostasis and stress response changed their expression abundances at various Ca levels. All the identified antioxidant enzymes regulating redox homeostasis showed increased or equal expression abundance at low Ca level compared with the control, including APX (spot 52), cytosolic APX (spot 57), chloroplast stromal APX (spot 58), tau class glutathione S-transferase (GST, spot 59), members of SOD (spot 60-62) and 2-Cys peroxiredoxin BAS1 (spot 66). The higher expression of antioxidant enzymes in plants induced by various abiotic/biotic stresses has been widely reviewed; the up-regulated enzymes enable plant to strengthen stress tolerance and resistance through maintaining redox homeostasis.54,55Ca deficiency is a kind of nutrient deficiency stress, and our study hints Ca deficiency may share some common oxidative damage patterns with other abiotic/biotic stresses that cause plant growth inhibition. However, the total APX activity and the total SOD activity showed declined trend at the low Ca level (Figure 1H and I), which looks contradictory with the increased APX and SOD expression. It can be interpreted as follow. Under long time (two months) Ca deficiency stress in our study, the translation of antioxidant enzymes were activated and contributed to accumulated enzymes. Nonetheless, the overall ability of these enzymes to respond oxidative stress may decline gradually with the elongation of stress time, these enzymes showed decreased activity and even deactivated completely as a result of long time Ca deficiency stress.
In the high Ca level, the majority of these antioxidant enzymes showed declined expression. The rationalization to this phenomenon is that adequate exogenous Ca supply relived Ca deficiency stress and subsequent oxidative stress, therefore there is no necessity to sustain high level of antioxidant enzymes.
What’s interesting is that cytosolic APX (spot 57), chloroplast stromal APX (spot 58) and Mn-SOD (spot 62) showed increased abundance at both low Ca level and high Ca level compared with the control. Low Ca treatment induced higher APX and SOD expression may probably due to the Ca deficiency resulted oxidative stress. While the up-regulated cytosolic APX, chloroplast stromal APX and Mn-SOD at high Ca level probably be related to the exogenous Ca improved P. massonianaseedling photosynthesis (Figure 1B and C). In the process of photosynthetic electron transfer and enzymatic reaction, it is inevitable to produce some by-products such as reactive oxygen species (ROS) including superoxide anion radical (O2ˉ) and hydrogen peroxide (H2O2), while H2O2 production even acts as an index for photosynthetic electron transport activity evaluation.56,57Strengthened photosynthesis signifies accelerated ROS production and accumulation.57 Within plant cell, especially in chloroplast, O2ˉ is catalyzed to H2O2 by SOD, and then H2O2 is reduced to water by APX.46,47SOD and APX play critical roles in photosynthesis by regulating ROS level.57,58High Ca treatment induced the up-regulation of cytosolic APX, chloroplast stromal APX and Mn-SOD manifests exogenous Ca activated the APX and SOD to remove excessive ROS, especially O2ˉ and H2O2, and then improved photosynthesis. The interaction between APX and SOD in the PPI network (Figure 5) further proved their collaborative role in ROS elimination. The increased H2O2 content, total APX activity and total SOD activity (Figure 1G-I) under high Ca treatment evidenced this deduction at physiological level.
Three stress response proteins, abscisic stress ripening protein 2 (spot 65), transcription factor bHLH145 (spot 48) and lipoxygenase (spot 5) showed similar changing patterns in expression abundances with that in antioxidant enzymes (Table 1). Transcription factor bHLH145 execute a positive regulation of stress response, such as drought and salinity stress response.59Lipoxygenase initiate the hydroperoxidation process of polyunsaturated fatty acids and resulted the formation of a variety of oxylipins, such as plant hormone jasmonic acid60. Various stresses could activate the expression of lipoxygenase and motivate the sequential stress response pathways.60 Abscisic stress ripening protein 2 showed increased abundance under drought stress.61 Compared with low Ca treatment, the down-regulation of the three proteins at high Ca level further proved adequate exogenous Ca can alleviate ROS stress and confer P. massoniana seedlings advanced environment adaptability.